Mindin基因巨噬细胞特异性敲除小鼠的构建及其在肺缺血再灌注损伤中的作用机制

Construction and mechanism of Mindin gene macrophage-specific knockout mice in lung ischemia-reperfusion injury

目的:探讨Mindin基因巨噬细胞特异性敲除小鼠的构建,及其在肺缺血再灌注损伤(ischemia-reperfusion injury,IRI)中的作用机制。方法:通过Cre-Lop系统构建Mindin基因巨噬细胞内特异性敲除小鼠,将小鼠分为C57/B6野生型小鼠假手术组(Sham组,10只)、C57/B6小鼠手术组(Surgery组,10只)、C57/B6小鼠手术组+Mindin重组蛋白干预组[Surgery(WT)+Mindin组,10只]和Mindin-/-巨噬细胞特异性敲除小鼠手术组[Surgery(Mindin-/-)组,10只]。通过夹闭肺门法构建肺IRI模型,观察该基因敲除及重组蛋白干预后,对肺IRI诱导的急性肺损伤(acute lung injury,ALI)、相关炎症因子IL1β、IL-18、TNF-α、高迁移速率蛋白B1(HMGB1)及核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、Gasdermin-D蛋白(GSDMD)、整合素β4(Integrinβ4)的影响。同时,对小鼠巨噬细胞J774A细胞系进行干预,检测不同缺氧复氧分组(缺氧复氧组、缺氧复氧+Mindin重组蛋白组和缺氧复氧+Mindin siRNA组)中NLRP3、GSDMD及Integrinβ4蛋白的表达,及不同Mindin重组蛋白分组(重组蛋白组、Mindin重组蛋白+Vehicle组和Mindin重组蛋白+Integrinβ4敲除组)中NLRP3、GSDMD蛋白的表达。使用独立样本t检验以及单因素方差对研究结果进行统计学分析。结果:本研究成功构建Mindin基因在巨噬细胞内特异性敲除小鼠。Surgery(Mindin-/-)组与Surgery组相比,小鼠肺水肿减轻;炎症因子IL1β、IL-18、TNF-α、HMGB1的释放均减少(2.73±0.19比5.81±0.61,6.52±0.63比11.03±0.34,2.18±0.14比4.76±0.20,14.57±0.33比8.76±0.87),组间比较,差异均有统计学意义(P均<0.05);NLRP3、GSDMD、Integrinβ4分泌均下调(2.07±0.27比4.91±0.22,2.78±0.37比5.78±0.29,3.04±0.75比7.71±0.34),组间比较,差异亦有统计学意义(P均<0.05)。Surgery(WT)+Mindin重组蛋白组与Surgery组比较,上述相关指标结果均出现上调,组间比较,差异亦有统计学意义(P均<0.05)。小鼠巨噬细胞J774A细胞系缺氧复氧组、缺氧复氧+Mindin siRNA组和缺氧复氧+Mindin重组蛋白组NLRP3、GSDMD及Integrinβ4蛋白分别为1.00±0.36比0.41±0.06比4.13±0.23、1.00±0.17比0.34±0.16比6.32±0.46和1.00±0.11比0.28±0.07比3.53±0.17。与缺氧复氧组比较,缺氧复氧+Mindin重组蛋白组上述参数均上调,而缺氧复氧+Mindin siRNA组均下调,且组间差异均有统计学意义(P均<0.05)。Mindin重组蛋白组、Mindin重组蛋白+Vehicle组、Mindin重组蛋白+Integrinβ4敲除组中NLRP3和GSDMD蛋白分别为1.00±0.07比1.13±0.11比0.51±0.14和1.00±0.09比0.87±0.16比0.37±0.12。Mindin重组蛋白+Integrinβ4敲除组上述参数较Mindin重组蛋白组及Mindin重组蛋白+Vehicle组均下调,且组间差异均有统计学意义(P均<0.05)。结论:在肺IRI过程中Mindin敲除能够减轻IRI,Mindin基因可能通过激活整合素Integrinβ4,进而促进相关炎症因子、NLRP3、GSDMD焦亡蛋白的表达,加重细胞焦亡从而促进肺IRI的发生发展。

Objective To explore the construction and mechanism of Mindin gene specific macrophage knockout mice in acute lung injury induced by lung ischemia-reperfusion injury(IRI).Methods Mindin gene knockout mice were constructed by CRE-LOP system,Mice were divided into four groups of C57/B6 wild-type mice sham operation(n=10),C57/B6 mice operation(n=10),Mindin-/-macrophage-specific knockout mice operation(n=10)and C57/B6 mice operation+Mindin recombinant protein intervention(n=10).And lung ischemia-reperfusion injury model was established by clamping pulmonary portal.The effects of Mindin gene knockout and recombinant protein intervention on acute lung injury were observed in vivo and in vitro.t-test and ANOVA test were employed for data processing.Results Mindin gene macrophage specific knockout mice was successfully constructed.Surgery(Mindin-/-)group significantly reduced pulmonary edema,release of inflammatory factors(IL1β:2.73±0.19 vs.5.81±0.61;IL-18:6.52±0.63 vs.11.03±0.34;TNF-α2.18±0.14 vs.4.76±0.20;HMGB1:4.57±0.33 vs.8.76±0.87),expression of NLRP3(2.07±0.27 vs.4.91±0.22)and secretion of GSDMD(2.78±0.37 vs.5.78±0.29)as compared with surgery group in vivo.In surgery(WT)+Mindin group,the expression of lung IRI,inflammatory factors and cell pyroptosis were opposite,And the results were consistent in vitro and in vivo.As compared with surgery group,the above parameters were up-regulated in surgery(WT)+Mindin protein group.And inter-group differences were statistically significant(all P<0.05).In vitro,the expressions of NLRP3(1.00±0.36,0.41±0.06,4.13±0.23),GSDMD(1.00±0.17,0.34±0.16,6.32±0.46)and integrinβ4(1.00±0.11,0.28±0.07,3.53±0.17)were detected in different groups including hypoxia-recovery oxygen(HR),HR+Mindin siRNA and HR+Mindin protein groups in macrophage cell line(J774A);As compared with HR group,the above parameters were up-regulated in HR+Mindin protein group and down-regulated in HR+Mindin siRNA group.And the differences were statistically significant(P<0.05).The expressions of NLRP3(1.00±0.07,1.13±0.11,0.51±0.14)and GSDMD(1.00±0.09,0.87±0.16,0.37±0.12)were detected in Mindin,Mindin protein+vehicle and Mindin protein+integrinβ4 knockout groups.The above parameters were down-regulated in Mindin protein+integrinβ4 knockout group as compared with Mindin protein and Mindin protein+vehicle groups.And the inter-group differences were statistically significant(all P<0.05).Conclusions During pulmonary IRI,Mindin knockdown can alleviate pulmonary IRI.Mindin gene may promote the expression of inflammatory factors,NLRP3 and GSDMD protein by activating integrinβ4 and aggravate cell pyroptosis to promote the development of pulmonary IRI.